Actin subunits assemble into many different three-dimensional structures that are central to processes such as cellular division and motility, yet how a cell assembles different cytoskeletal structures using actin as the basic building block remains unclear. I hypothesize that tropomyosin isoforms determine which actin structures are assembled by regulating which accessory factors can gain access to actin filaments. This argument is supported by data that eukaryotic cells can express multiple different tropomyosin isoforms, where each isoform is sorted to a unique actin structure. Although mammalian tropomyosins are being characterized, the fact that mammalian non-muscle cells express over forty different tropomyosin isoforms has made tropomyosin research very complex. Our lab has chosen Drosophila melanogaster S2 cells as the model organism for understanding why eukaryotic non-muscle cells express multiple tropomyosin isoforms. The S2 cell line is the quintessential model system for the study of tropomyosins because S2 cells are easy to grow, are highly susceptible to gene inhibition using RNAi, have well-characterized actin networks, and most importantly, express only three tropomyosin isoforms in comparison to mammalian cells. Recent data from the Mullins lab discovered that, like mammalian cells, the Drosophila tropomyosin isoforms each localize to distinct actin structures in S2 cells. When these individual tropomyosins are mutated, the cell exhibits cell morphology and cell division abnormalities, suggesting that all three tropomyosin isoforms are required. The main objective of this project is to identify the functions of the three tropomyosin isoforms. In the first part of my proposal, I wil uncover the assembly mechanism of tropomyosin on actin filaments using a single filament Total Internal Reflection Fluorescence (TIRF) microscopy. Since the species the actin was purified from significantly influences tropomyosin activity, I will use only Drosophila-based components. In the second part of my proposal, I will identify the relationship between tropomyosins Tm1A and Tm1J and their relationship with myosin II and formin, respectively. I will use gliding assays to study myosin binding and activity and single filament TIRF microscopy to visualize the activity of formins and tropomyosins. This proposal will be the first to characterize every single tropomyosin informs for a given cell type, revealing the roles of tropomyosin isoforms in eukaryotic cells. In addition, understanding tropomyosins will also reveal how they assemble different actin structures.